Sclerotinia stem rot (SSR) is caused by the necrotrophic fungus Sclerotinia sclerotiorum and threatens global oilseed rape (Brassica napus) production. Moreover, researchers have not yet identified a gene that confers complete resistance. Here, we developed a multi-target RNA interference (RNAi) strategy to enhance plant resistance by simultaneously silencing eight fungal genes involved in development (SsChsI–VII, SsGas1) and two involved in pathogenicity (SsPG1, SsOAH1) of S. sclerotiorum. Accordingly, we designed a 1,250-bp chimeric double-stranded RNA (dsRNA) consisting of ten 125-bp fragments each targeting a different gene, and evaluated its effectiveness using spray-induced gene silencing (SIGS) and host-induced gene silencing (HIGS) via stable transformation. In vitro application of the chimeric dsRNA resulted in >50% downregulation of nine target genes, indicating efficient uptake and processing by S. sclerotiorum. Both lesion area and fungal biomass were significantly lower in Nicotiana benthamiana and oilseed rape plants following SIGS. Moreover, stable transgenic plants for HIGS effectively generated gene-specific short interfering RNAs and exhibited an increase in resistance from the T2 to T5 generations, with lesions that were 38.9–59.1% smaller in leaves and 43.2–65.8% smaller in stems in the T5 generation compared with the control plants. Gene silencing resulted in lower oxalic acid accumulation, decreased polygalacturonase activity, and impaired hyphal development, suggesting interference with multiple fungal infection pathways. Notably, HIGS conferred stable, heritable resistance without yield penalty, whereas SIGS provided rapid, nontransgenic protection. This study demonstrates the effectiveness of long chimeric dsRNAs for multi-target gene silencing and highlights a promising RNAi-based strategy for improving disease resistance in oilseed rape, possibly in combination with natural quantitative resistance loci.

Klebsiella pneumoniae (K. pneumoniae, KP) is a significant opportunistic pathogen responsible for both nosocomial and community-acquired infections. Bacterial adhesion is the critical initial step for host colonization and the establishment of disease. In this study, we utilized a mariner transposon mutagenesis system to construct a mutant library from the clinical KP strain KP20, identifying a mutant with significantly impaired epithelial cell adhesion due to an insertion in the uspF gene. Genetic knockout experiments confirmed that uspF deletion markedly reduced the adhesion to human airway epithelial cells (Calu-3) and downregulated the transcription of type III pili-encoding genes (mrkABDF). Furthermore, uspF deficiency compromised antioxidant stress and serum resistance and increased susceptibility to dendritic cell and macrophage phagocytosis. In vivo challenge experiments further demonstrated that uspF deletion significantly attenuated K. pneumoniae virulence in mice. These findings provide important insights into the molecular pathogenesis of K. pneumoniae and identify UspF as a potential target for therapeutic intervention.

Antibiotic misuse accelerates resistance dissemination via plasmid conjugation, but quorum sensing (QS) regulatory mechanisms remain undefined. Using Escherichia coli (E. coli) MG1655 conjugation models (RP4-7/EC600 plasmids), we demonstrate that long-chain acyl-homoserine lactones (C10/C12-HSL) enhance transfer frequency by up to 7.7-fold (200μM C12-HSL; p < 0.001), while quorum-quenching by sub-inhibitory vanillin suppressed this effect by 95% (p < 0.0001). C12-HSL compromised membrane integrity via ompF upregulation (4-fold; p < 0.01) and conjugative pore assembly (trbBp upregulated by 1.38-fold; p < 0.05), coinciding with ROS accumulation (1.5-fold; p < 0.0001) and SOS response activation (recA upregulated by 1.68-fold; p < 0.001). Crucially, rpoS and rmf deletion mutants reduced conjugation by 65.5% and 55.8%, respectively (p < 0.001), exhibiting attenuated membrane permeability (≤65.5% reduced NPN influx; p < 0.0001), suppressed ROS (≤54% downregulated; p < 0.0001), and abolished transcriptional induction of conjugation/stress genes. Reciprocal RpoS–RMF (ribosomal hibernation factor) crosstalk was essential for AHL responsiveness, with deletions mutually suppressing expression (≤65.9% downregulated; p < 0.05). We establish a hierarchical mechanism wherein long-chain AHLs drive resistance dissemination through integrated membrane restructuring, stress adaptation, and RpoS–RMF-mediated genetic plasticity, positioning QS signaling as a viable target for curbing resistance spread.

Klebsiella pneumoniae, a zoonotic pathogen of global concern, poses significant threats to both veterinary and public health. Here, a comparative study characterized 14 clinical isolates (7 avian-derived, 7 human-derived) from Jiangsu, China, through integrated genomic and phenotypic analyses. Firstly, multilocus sequence typing (MLST) revealed distinct epidemiological patterns: the same ST type in avian isolates was circulating between different species and different regions, whereas it was not found in human isolates. In addition, hypervirulent Klebsiella pneumoniae (hvKP) phenotypes confirmed by string test were exclusive to two human isolates (KP15, KP20). Secondly, biofilm detection demonstrated 78.6% (11/14) of isolates possessed biofilm-forming capacity, with cellulose but not curli as the predominant matrix component. Human-derived KP15 and KP20 had the strongest biofilm formation ability in all isolates. Antimicrobial susceptibility profiling identified serious multidrug resistance in both avian and human isolates. Virulence gene analysis revealed striking disparities, with human isolates harboring 10–20 virulence factors (median 15) versus 6–7 (median 6.5) in avian counterparts. Finally, functional pathogenesis assessments demonstrated human-derived strains exhibited stronger epithelial cell adhesion (2-fold higher) and invasion (1.97-fold higher) in Calu-3 cell models and paradoxically showed reduced macrophage phagocytosis (2.85-fold lower at 2 h) for immune escape. In vivo models confirmed dose-dependent mortality, with human isolates demonstrating higher lethality in both Galleria mellonella and mice. Virulence gene burden positively correlated with mortality outcomes. These findings delineate critical host adaptation differences in Klebsiella pneumoniae populations and provide empirical evidence for pathogen transmission dynamics at the human-animal interface.

The mechanism by which quorum sensing (QS) enhances stress resistance in enterohemorrhagic Escherichia coli (E. coli) O157:H7 remains unclear. We employed optimized exogenous QS signal N-acyl-homoserinelactones (AHL) (100 μM 3-oxo-C6-AHL, 2 h) in EHEC O157:H7 strain EDL933, which was validated with endogenous yenI-derived AHL, to investigate QS-mediated protection against acid stress. RNA-seq transcriptomics identified key upregulated genes (e.g., rmf). Functional validation using isogenic rmf knockout mutants generated via λ-Red demonstrated abolished stress resistance and pan-stress vulnerability. Mechanistic studies employing qRT-PCR and stress survival assays established Ribosomal Hibernation Factor (RMF) as a non-redundant executor in a SdiA–RMF–RpoS axis, which activates ribosomal dormancy and SOS response to enhance EHEC survival under diverse stresses. For the first time, we define ribosomal hibernation as the core adaptive strategy linking QS to pathogen resilience, providing crucial mechanistic insights for developing EHEC control measures against foodborne threats.

Quorum sensing (QS) is a process by which bacteria sense their population density and regulate behavior accordingly. QS not only regulates bacterial virulence but also directly influences host cells. Previous studies have shown that QS is strongly associated with piglet intestinal health, but the mechanism is not yet clear. For the first time, we have confirmed in a piglet animal model that OdDHL directly damages intestinal cells in weaned piglets, disrupting the intestinal barrier. We also provide a preliminary exploration of the underlying mechanism of these effects. TUNEL assays confirmed that damage to the piglet intestinal barrier coincided temporally and spatially with dysregulated apoptosis. Lipid rafts, key components of the cell membrane, are involved in many biological processes, including the activation of apoptosis-related proteins. Following the disruption of the lipid raft structure in IPEC-J2 cells, the apoptosis rate under OdDHL stimulation decreased by 50%. These data demonstrate that lipid rafts mediate the attachment of OdDHL to porcine intestinal cells; then, OdDHL induces apoptosis in porcine intestinal cells through the mitochondrial and death receptor pathways, thereby compromising the integrity of the porcine intestinal barrier. This study provides foundational insights into the role of QS in piglet intestinal diseases.

鞭毛是位于细菌表面的长螺旋形可旋转附属物,长约10 μm,主要与细菌的运动有关.鞭毛的驱动力是许多细菌病原体的重要毒力特征,并且是建立感染所必需的.感染发生后,鞭毛有利于细菌到达侵入部位.大肠杆菌(Escherichia coli,E.coli)一般周身鞭毛,具有运动性.鞭毛蛋白,又称为H抗原,是大肠杆菌分类的重要表面抗原.本文对大肠杆菌鞭毛的结构、功能和H抗原分型作一简要综述,旨在为本领域的相关研究提供一定的理论基础和依据.

恶臭假单胞菌是一种广泛存在于环境中的人兽共患条件致病菌,主要引起水生动物疾病,其对禽类的致病性研究目前没有引起关注.本研究从临床上78日龄脚软病鸡的跖趾关节腔中分离出1株病原菌,结合临床症状、细菌培养和血清学凝集反应,前期一直怀疑为沙门菌,通过进一步的微生物质谱检测、生化反应和16S rDNA测序,确定为恶臭假单胞菌.药敏试验结果显示该分离株对头孢曲松、头孢他啶、庆大霉素、丁胺卡那、新霉素、四环素和环丙沙星7种受试抗菌药物敏感,对链霉素、青霉素、呋喃唑酮、磺胺异噁唑、氯霉素、氟苯尼考、林可霉素、红霉素、恩诺沙星、诺氟沙星10种受试抗菌药物均耐药.本研究首例报道从软脚病鸡临床分离到恶臭假单胞菌,对兽医临床鉴别诊断恶臭假单胞菌感染及临床防治用药具有指导意义和参考价值.

产肠毒素性大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是引起婴幼儿和幼畜腹泻的主要细菌性病原,初生幼畜感染后常因剧烈腹泻脱水而死亡.菌毛黏附素和肠毒素是ETEC目前研究最多的2类毒力因子,随着技术手段的不断进步和研究的深入,一些新型毒力因子不断被发现,如鞭毛、非菌毛黏附素、肠聚集性耐热毒素(the enteroaggregative heat-stable toxin1,EAST1)、自转运黏附蛋白、细胞溶血素等.本综述主要针对猪源ETEC的主要毒力因子以及新型毒力的结构功能及致病性进行阐述,为进一步深入探索ETEC的致病机理提供理论基础.

Outer membrane vesicles (OMVs) are promising vaccine components because they combine antigen and adjuvant in a single formulation. Detoxified Salmonella enterica strains that express penta-acylated lipid A retain OMV immunogenicity but with reduced reactogenicity. We have previously shown that a recombinant form of the enterotoxigenic Escherichia coli (ETEC) 17 kilodalton protein (Skp) protects mice in a pulmonary challenge model, when fused to the glutathione-S-transferase (GST) epitope and combined with cholera toxin. Here we compared directly the efficacy of expressing Skp in detoxified Salmonella OMVs to GST-Skp for their ability to protect mice against ETEC challenge. We observed that the display of Skp on OMVs, in the absence of exogenous adjuvant, protects the mice as well as the recombinant GST-Skp with adjuvant, showing that we can achieve protection when antigen and adjuvant are administered as a single formulation. Collectively, these data demonstrate the utility of using OMVs for the expression and display of antigens for use in vaccine development and validate previously published work demonstrating that immunization with Skp is efficacious in protecting mice against ETEC challenge.

为进一步探析大肠杆菌Ⅰ型群体感应系统(QS-Ⅰ)对生物被膜(BF)形成能力的影响,研究构建可合成内源性QS-Ⅰ酰基高丝氨酸内酯(AHL)的大肠杆菌,模拟自然界中禽致病性大肠杆菌(APEC)受外源AHL调控的独特现象,分别通过玻璃试管气液交界面壁、聚苯乙稀材料表面、玻璃平面等不同环境下生物被膜形成能力,检测QS-Ⅰ对APEC BF形成能力的调控,并通过荧光定量PCR的方法检测相关基因,对调控机制进行初步探讨.结果显示,QS-Ⅰ系统抑制APEC菌株的生物被膜形成能力,且BF的形成不受鞭毛因素影响,APEC菌株可能通过QS-Ⅰ对QS-Ⅱ的抑制来间接调控BF的形成.研究结果提供了研究APEC毒力调控的新视角.

H9N2 avian influenza virus (AIV) evolves rapidly in both genovariation and antigenicity. It is essential to monitor the change of antigenicity, in particular in the hemagglutinin (HA) protein. Here we reported the selection of antigenic variants from A/Chicken/Shanghai/F/98 (H9N2) and A/chicken/Taixing/10/2010 (H9N2) viruses using HA-specific monoclonal antibodies (MAbs). Based on the reactivity of these variant and wild-type strains with the MAbs, we identified 6 critical amino acid positions (92, 145, 166, 167, 168, and 197) in the H9 antigenic sites, including the position 92 that has never been reported. Among AIVs originated from chicken in mainland China, the rates of Gly and Arg at position 92 within BJ/94-like (A/chicken/Beijing/1/1994) lineage viruses were 62.2% (28/45) and 37.8% (17/45), respectively; whereas the rates of Gly and Arg at position 92 within Y280-like (A/duck/Hong Kong/Y280/97) lineage viruses were 0.3% (2/670) and 99.1% (673/679), respectively. Our study suggests that G92R mutation together with other identified antigenic sites may serve as molecular markers for H9N2 virus evolution, and may aid improving AIV vaccine effectiveness.

Bacterial flagella contribute to pathogen virulence; however, the role of flagella in the pathogenesis of F18ab E. coli-mediated swine edema disease (ED) is not currently known. We therefore evaluated the role of flagella in F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production using an in vitro cell infection model approach with gene-deletion mutant and complemented bacterial strains. We demonstrated that the flagellin-deficient fliC mutant had a marked decrease in the ability to adhere to and invade porcine epithelial IPEC-J2 cells. Surprisingly, there was no difference in adhesion between the F18 fimbriae-deficient ΔfedA mutant and its parent strain. In addition, both the ΔfedA and double ΔfliCΔfedA mutants exhibited an increased ability to invade IPEC-J2 cells compared to the wild-type strain, although this may be due to increased expression of other adhesins following the loss of F18ab fimbriae and flagella. Compared to the wild-type strain, the ΔfliC mutant showed significantly reduced ability to form biofilm, whereas the ΔfedA mutant increased biofilm formation. Although ΔfliC, ΔfedA, and ΔfliCΔfedA mutants had a reduced ability to stimulate IL-8 production from infected Caco-2 cells, the ΔfliC mutant impaired this ability to a greater extent than the ΔfedA mutant. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro.